The invention relates to a method for the kinetic determination of the concentration of a dissolved or suspended substrate, wherein at least one enzyme is added and the speed of the enzymatic reaction is measured. The method is particularly suitable for the quantitative determination of substrates in blood serum or blood plasma. The invention furthermore concerns reagents for the practice of the method. The importance of the invention lies especially in the fields of clinical chemistry, biochemistry, food chemistry and pharmacy.
Especially in the analysis of biological liquids of complex composition, enzymatically catalyzed reactions are preferred for their substrate specificity in the reaction and for their high sensitivity.
On a routine basis, determinations of substrate concentration by means of enzymatically catalyzed reactions have hitherto usually been performed as end-value determinations, wherein a parameter is measured before and after the reaction to establish the difference between them.
The determination of uric acid by the Kageyama method, in which two enzymes are added, is an exception. What is kinetically measured, however, is the slow, nonenzymatic part of the action, so that this is basically different method. This known method takes a considerable amount of time, since measurements must be made after 6 and 12 minutes. This method is therefore expensive, inasmuch as an elevated temperature of 37.degree.C has to be maintained. It would be advantageous if it were possible to perform this determination at the 25.degree.C temperature usually used for the kinetic determination of enzyme activities, and thus to perform it with the apparatus already on hand.
Another exception is the determination of glucose with glucose oxidase, which can be performed kinetically with routinely used photometers, because the value of the Michaelis constant is exceptionally good for this reaction.
A kinetic determination of substrate concentrations, in which the change of a parameter is measured in the course of a reaction, offers important advantages over end-value determinations:
1. The amount of time required is shorter than in the corresponding end-value method. PA1 2. A specimen blank value can usually be dispensed with, thereby reducing the number of steps required for the preparation of test solutions, reducing the consumption of specimen material and reagents, and in some cases reducing apparatus tie-up and on-line expense when computers are used. PA1 3. Substrate determination can be performed with the apparatus commonly used for determining enzyme activity. This also makes possible the simultaneous performance of determinations which have hitherto been difficult to combine due to apparatus limitations. PA1 4. The use of disposable cells, which eliminates rinsing and precludes contamination erros in the photometric measurements, is possible without restriction, because errors cannot occur due to differences in the inherent extinction of the cells. PA1 1. Extremely precise, expensive measuring instruments of high resolution are required, because the meter deflection in the case of very small substrate transformations is within the range of fluctuation of conventional apparatus such as photometers, for example. PA1 2. Only the low portion of the concentration spectrum of the specimens can be tested without preliminary dilution; preliminary dilution is a source of additional errors and necessitates repetition of the analysis. PA1 3. Dirt particles and air bubbles can greatly falsify the readings; in routine operations the necessary extreme cleanliness cannot always be assured. PA1 a. Substances which (in accordance with the law of mass action) compete reversibly with the substrate for the enzyme, but are transformed not at all or with negligible slowness (competitive inhibitors in the conventional sense). PA1 b. Substances which reversibly bind the substrate and thereby diminish its free concentration (in accordance with the law of mass action), the inhibitor-substrate complex not being transformed by the effect of the enzyme. PA1 c.sub.SI = concentration of the substrate-inhibitor complex, PA1 c.sub.S = concentration of the free substrate, PA1 C.sub.EI = concentration of the enzyme-inhibitor complex, PA1 c.sub.E = free enzyme concentration.
In spite of all these obvious advantages, kinetic substrate determinations have not been routinely practiced and can be performed rationally only in exceptional cases. The known enzyme-kinetic techniques ("initial rate" and "time-fixed" techniques) are important only in research; they have it in common that only very small substrate concentration can be used and hence only very small substrate transformations can be measured.
The limitation to very small substrate concentrations in the test solution is necessary because only thus will there be a direct proportionality between the concentration of the substrate being tested and the speed of the reaction, i.e., only thus will a linear calibration curve be obtained, which is an essential requirement for simple routine determinations.
That is to say, the Michaelis-Menten equation ##EQU1## applies to the speed of enzymatically catalyzed reactions (v = speed of reaction; v.sub.max = maximum speed in the substrate saturation range; c.sub.S = substrate concentration; K.sub.M = the Michaelis constant).
On the basis of this equation, proportionality will exist between the reaction speed v and the substrate concentration c.sub.S only when c.sub.S is very small in relation to K.sub.M. The term c.sub.S in the numerator can then be neglected, so that kinetically the result is a reaction of the pseudo-first order. As theoretical considerations show, c.sub.S may not be greater than 0.2 K.sub.M in order to keep the analytic error within acceptable limits for kinetic determinations.
Now, for most reactions, K.sub.M itself is very small, being of the order of magnitude of 10.sup.-.sup.3 to 10.sup.-.sup.7 moles per liter. It will be seen from this that measurements by the methods of the prior art are limited to extremely small substrate concentrations.
Attempts have been made to counter this difficulty by using enzymes having the highest possible value of K.sub.M. The available selection, however, is small, and even in the most favorable case, the K.sub.M values are as a rule too low for substrate determinations in actual practice (the determination of glucose with GOD is one exception, as mentioned above).
From what has been stated above, the known kinetic enzyme methods suffer from the following disadvantages: